Insulin increases active sodium transport across isolated toad skin segments, and this response can be directly and continuously monitored by measuring the short circuit current. In this project it is intended to study tissue-binding and degradation of insulin by the toad skin preparation. It is also intended to evaluate the possible role of adenyl cyclase in the stimulatory action of insulin in this tissue. Preliminary studies showed: 1) graded responses to insulin concentrations varying from 10 to the minus 9th power M to 10 to the minus 7th power M; 2) similar responses with respect to time of onset and maximal effect, whether skins were exposed continuously or as briefly as 30 sec to insulin 10 to minus 7th power M; 3) rapid disappearance of immunoreactive and TCA precipitable insulin from the incubation medium; 4) impairment of responsiveness to insulin and vasopressin following exposure of skins to trypsin (1 mg/ml) for 1 hr, but normal response to the stimulatory effect of theophylline. It is intended to perform the following additional studies: 1) determination of the effect of enzymes and other agents that alter cell membrane surface upon responsiveness of the insulin by isolated cell preparations; 2) direct measurements of specific binding of I125-insulin by isolated toad skin epithelial cells; 3) measurement of cyclic AMP concentrations and activities of adenyl cyclase and cyclic AMP phosphodiesterase by isolated epithelial cells in the presence and absence of insulin; 4) the effects of controlled proteolysis upon these activities.